More than a colour; how pigment influences colourblind microbes.

Many animals have pigments when they themselves cannot see colour. Perhaps those pigments enable the animal to avoid predators, or to attract mates. Maybe even those pigmented surfaces are hosts for microbes, even when the microbes do not see colour. Do some pigments then serve as a chemical signal for a good or bad microbial substrate? Maybe pigments attract or repel various microbe types? Echinoderms serve as an important model to test the mechanisms of pigment-based microbial interactions. Echinoderms are marine benthic organisms, ranging from intertidal habitats to depths of thousands of metres and are exposed to large varieties of microbes. They are also highly pigmented, with a diverse variety of colours between and even within species. Here we focus on one type of pigment (naphthoquinones) made by polyketide synthase, modified by flavin-dependent monoxygenases, and on one type of function, microbial interaction. Recent successes in targeted gene inactivation by CRISPR/Cas9 in sea urchins supports the contention that colour is more than it seems. Here we dissect the players, and their interactions to better understand how such host factors influence a microbial colonization. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Single-Cell Transcriptome and Pigment Biochemistry Analysis Reveals the Potential for the High Nutritional and Medicinal Value of Purple Sea Cucumbers.

The sea cucumber Apostichopus japonicus has important nutritional and medicinal value. Unfortunately, we know little of the source of active chemicals in this animal, but the plentiful pigments of these animals are thought to function in intriguing ways for translation into clinical and food chemistry usage. Here, we found key cell groups with the gene activity predicted for the color morphology of sea cucumber body using single-cell RNA-seq. We refer to these cell populations as melanocytes and quinocytes, which are responsible for the synthesis of melanin and quinone pigments, respectively. We integrated analysis of pigment biochemistry with the transcript profiles to illuminate the molecular mechanisms regulating distinct pigment formation in echinoderms. In concert with the correlated pigment analysis from each color morph, this study expands our understanding of medically important pigment production, as well as the genetic mechanisms for color morphs, and provides deep datasets for exploring advancements in the fields of bioactives and nutraceuticals.

Molecular mechanisms of tubulogenesis revealed in the sea star hydro-vascular organ.

A fundamental goal in the organogenesis field is to understand how cells organize into tubular shapes. Toward this aim, we have established the hydro-vascular organ in the sea star Patiria miniata as a model for tubulogenesis. In this animal, bilateral tubes grow out from the tip of the developing gut, and precisely extend to specific sites in the larva. This growth involves cell migration coupled with mitosis in distinct zones. Cell proliferation requires FGF signaling, whereas the three-dimensional orientation of the organ depends on Wnt signaling. Specification and maintenance of tube cell fate requires Delta/Notch signaling. Moreover, we identify target genes of the FGF pathway that contribute to tube morphology, revealing molecular mechanisms for tube outgrowth. Finally, we report that FGF activates the Six1/2 transcription factor, which serves as an evolutionarily ancient regulator of branching morphogenesis. This study uncovers distinct mechanisms of tubulogenesis in vivo and we propose that cellular dynamics in the sea star hydro-vascular organ represents a key comparison for understanding the evolution of vertebrate organs.

Elements of divergence in germline determination in closely related species.

Evolutionary transitions are particularly important in development of the germ line, cells which directly impact sexual reproduction. Differences in the primordial germ cells (PGCs) of two sea urchin species were examined here by stage-matched, integrated, single cell RNA-seq (scRNA-seq) datasets. Even though both species rely on inherited mechanisms to specify their germ line, this analysis revealed a variety of differences in germline gene expression, including a broader expression of the germline factor Nanos2 (Nan2) in Lytechinus variegatus (Lv) compared to Strongylocentrotus purpuratus (Sp). In Sp, Nan2 mRNA expression is highly restricted to the PGCs by a lability element in its 3'UTR, which is lacking in the mRNA of Lv-Nan2, thus explaining the difference. We discovered that the Lv-Nan2 3'UTR instead leads to its specific translation in the PGCs. The results emphasize that regulatory mechanisms resulting in germline specification rely greatly on post-transcriptional restrictions of key gene products.

A case of hermaphroditism in the gonochoristic sea urchin, Strongylocentrotus purpuratus, reveals key mechanisms of sex determination†.

Sea urchins are usually gonochoristic, with all of their five gonads either testes or ovaries. Here, we report an unusual case of hermaphroditism in the purple sea urchin, Strongylocentrotus purpuratus. The hermaphrodite is self-fertile, and one of the gonads is an ovotestis; it is largely an ovary with a small segment containing fully mature sperm. Molecular analysis demonstrated that each gonad producedviable gametes, and we identified for the first time a somatic sex-specific marker in this phylum: Doublesex and mab-3 related transcription factor 1 (DMRT1). This finding also enabled us to analyze the somatic tissues of the hermaphrodite, and we found that the oral tissues (including gut) were out of register with the aboral tissues (including tube feet) enabling a genetic lineage analysis. Results from this study support a genetic basis of sex determination in sea urchins, the viability of hermaphroditism, and distinguish gonad determination from somatic tissue organization in the adult.

A Review of Asteroid Biology in the Context of Sea Star Wasting: Possible Causes and Consequences.

AbstractSea star wasting-marked in a variety of sea star species as varying degrees of skin lesions followed by disintegration-recently caused one of the largest marine die-offs ever recorded on the west coast of North America, killing billions of sea stars. Despite the important ramifications this mortality had for coastal benthic ecosystems, such as increased abundance of prey, little is known about the causes of the disease or the mechanisms of its progression. Although there have been studies indicating a range of causal mechanisms, including viruses and environmental effects, the broad spatial and depth range of affected populations leaves many questions remaining about either infectious or non-infectious mechanisms. Wasting appears to start with degradation of mutable connective tissue in the body wall, leading to disintegration of the epidermis. Here, we briefly review basic sea star biology in the context of sea star wasting and present our current knowledge and hypotheses related to the symptoms, the microbiome, the viruses, and the associated environmental stressors. We also highlight throughout the article knowledge gaps and the data needed to better understand sea star wasting mechanistically, its causes, and potential management.

Pigmentation biosynthesis influences the microbiome in sea urchins.

Organisms living on the seafloor are subject to encrustations by a wide variety of animals, plants and microbes. Sea urchins, however, thwart this covering. Despite having a sophisticated immune system, there is no clear molecular mechanism that allows sea urchins to remain free of epibiotic microorganisms. Here, we test the hypothesis that pigmentation biosynthesis in sea urchin spines influences their interactions with microbes in vivo using CRISPR/Cas9. We report three primary findings. First, the microbiome of sea urchin spines is species-specific and much of this community is lost in captivity. Second, different colour morphs associate with bacterial communities that are similar in taxonomic composition, diversity and evenness. Lastly, loss of the pigmentation biosynthesis genes polyketide synthase and flavin-dependent monooxygenase induces a shift in which bacterial taxa colonize sea urchin spines. Therefore, our results are consistent with the hypothesis that host pigmentation biosynthesis can, but may not always, influence the microbiome in sea urchin spines.

Optimizing CRISPR/Cas9-based gene manipulation in echinoderms.

The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of morpholino antisense oligonucleotide (MASO) technologies are now able to apply CRISPR-Cas9 to study biological problems in their model organism of choice much more effectively. The transitions to new CRISPR-based approaches could be enhanced, first, by standardized protocols and education in their applications. Here we summarize our results for optimizing the CRISPR-Cas9 technology in a sea urchin and a sea star, and provide advice on how to set up CRISPR-Cas9 experiments and interpret the results in echinoderms. Our goal through these protocols and sharing examples of success by other labs is to lower the activation barrier so that more laboratories can apply CRISPR-Cas9 technologies in these important animals.

Post-transcriptional regulation of factors important for the germ line.

Echinoderms are a major model system for many general aspects of biology, including mechanisms of gene regulation. Analysis of transcriptional regulation (Gene regulatory networks, direct DNA-binding of proteins to specific cis-elements, and transgenesis) has contributed to our understanding of how an embryo works. This chapter looks at post-transcriptional gene regulation in the context of how the primordial germ cells are formed, and how the factors essential for this process are regulated. Important in echinoderms, as in many embryos, is that key steps of fate determination are made post-transcriptionally. This chapter highlights these steps uncovered in sea urchins and sea stars, and links them to a general theme of how the germ line may regulate its fate differently than many of the embryo's somatic cell lineages.

A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos.

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies.

A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification.

Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.

Minor Genetic Consequences of a Major Mass Mortality: Short-Term Effects in Pisaster ochraceus.

AbstractMass mortality events are increasing globally in frequency and magnitude, largely as a result of human-induced change. The effects of these mass mortality events, in both the long and short term, are of imminent concern because of their ecosystem impacts. Genomic data can be used to reveal some of the population-level changes associated with mass mortality events. Here, we use reduced-representation sequencing to identify potential short-term genetic impacts of a mass mortality event associated with a sea star wasting outbreak. We tested for changes in the population for genetic differentiation, diversity, and effective population size between pre-sea star wasting and post-sea star wasting populations of Pisaster ochraceus-a species that suffered high sea star wasting-associated mortality (75%-100% at 80% of sites). We detected no significant population-based genetic differentiation over the spatial scale sampled; however, the post-sea star wasting population tended toward more differentiation across sites than the pre-sea star wasting population. Genetic estimates of effective population size did not detectably change, consistent with theoretical expectations; however, rare alleles were lost. While we were unable to detect significant population-based genetic differentiation or changes in effective population size over this short time period, the genetic burden of this mass mortality event may be borne by future generations, unless widespread recruitment mitigates the population decline. Prior results from P. ochraceus indicated that natural selection played a role in altering allele frequencies following this mass mortality event. In addition to the role of selection found in a previous study on the genomic impacts of sea star wasting on P. ochraceus, our current study highlights the potential role the stochastic loss of many individuals plays in altering how genetic variation is structured across the landscape. Future genetic monitoring is needed to determine long-term genetic impacts in this long-lived species. Given the increased frequency of mass mortality events, it is important to implement demographic and genetic monitoring strategies that capture baselines and background dynamics to better contextualize species' responses to large perturbations.

Is It in the Stars? Exploring the Relationships between Species' Traits and Sea Star Wasting Disease.

AbstractAn explanation for variation in impacts of sea star wasting disease across asteroid species remains elusive. Although various traits have been suggested to play a potential role in sea star wasting susceptibility, currently we lack a thorough comparison that explores how life-history and natural history traits shape responses to mass mortality across diverse asteroid taxa. To explore how asteroid traits may relate to sea star wasting, using available data and recognizing the potential for biological correlations to be driven by phylogeny, we generated a supertree, tested traits for phylogenetic association, and evaluated associations between traits and sea star wasting impact. Our analyses show no evidence for a phylogenetic association with sea star wasting impact, but there does appear to be phylogenetic association for a subset of asteroid life-history traits, including diet, substrate, and reproductive season. We found no relationship between sea star wasting and developmental mode, diet, pelagic larval duration, or substrate but did find a relationship with minimum depth, reproductive season, and rugosity (or surface complexity). Species with the greatest sea star wasting impacts tend to have shallower minimum depth distributions, they tend to have their median reproductive period 1.5 months earlier, and they tend to have higher rugosities relative to species less affected by sea star wasting. Fully understanding sea star wasting remains challenging, in part because dramatic gaps still exist in our understanding of the basic biology and phylogeny of asteroids. Future studies would benefit from a more robust phylogenetic understanding of sea stars, as well as leveraging intra- and interspecific comparative transcriptomics and genomics to elucidate the molecular pathways responding to sea star wasting.

Prediction of Genes That Function in Methanogenesis and CO2 Pathways in Extremophiles.

Gaet'ale (GAL) and Mud'ara (MUP) are two hypersaline ponds located in the Danakil Depression recharged by underground water from the surrounding highlands. These two ponds have different pH, salinity, and show variation in the concentration of many ionic components. Metagenomic analysis concludes that GAL is dominated by bacteria as in the case of the other hypersaline and acidic ponds in the Danakil Depression. However, Archaea dominated the ponds of MUP. In the current study, the application of SEED and KEGG helped to map the ordered steps of specific enzyme catalyzed reaction in converting CO2 into cell products. We predict that highly efficient and light-independent carbon fixation involving phosphoenolpyruvate carboxylase takes place in MUP. On the contrary, genes encoding enzymes involved in hydrogenotrophic and acetoclastic methanogenesis appeared solely in ponds of GAL, implying the biological source of the hazardous methane gas in that environment. Based on the investigation of the sources of the genes of interest, it is clear that cooperative interactions between members of the two communities and syntrophic metabolism is the main strategy adapted to utilize inorganic carbon as a carbon source in both MUP and GAL. This insight can be used to design biotechnological applications of microbial communities in production of methane biogas or to minimize CO2 emissions.

Sperm lacking Bindin are infertile but are otherwise indistinguishable from wildtype sperm.

Cell-cell fusion is limited to only a few cell types in the body of most organisms and sperm and eggs are paradigmatic in this process. The specialized cellular mechanism of fertilization includes the timely exposure of gamete-specific interaction proteins by the sperm as it approaches the egg. Bindin in sea urchin sperm is one such gamete interaction protein and it enables species-specific interaction with a homotypic egg. We recently showed that Bindin is essential for fertilization by use of Cas9 targeted gene inactivation in the sea urchin, Hemicentrotus pulcherrimus. Here we show phenotypic details of Bindin-minus sperm. Sperm lacking Bindin do not bind to nor fertilize eggs at even high concentrations, yet they otherwise have wildtype morphology and function. These features include head shape, tail length and beating frequency, an acrosomal vesicle, a nuclear fossa, and they undergo an acrosomal reaction. The only phenotypic differences between wildtype and Bindin-minus sperm identified is that Bindin-minus sperm have a slightly shorter head, likely as a result of an acrosome lacking Bindin. These data, and the observation that Bindin-minus embryos develop normally and metamorphose into normal functioning adults, support the contention that Bindin functions are limited to species-specific sperm-egg interactions. We conclude that the evolutionary divergence of Bindin is not constrained by any other biological roles.

Polarized Dishevelled dissolution and reassembly drives embryonic axis specification in sea star oocytes.

The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes.1 However, the mechanisms that generate cellular asymmetries within the oocyte and then transduce that polarity to organismal scale body axes are poorly understood outside of select model organisms. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled (Dvl) is a cytoplasmic Wnt pathway effector required for axis development in diverse species,2-4 but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dvl localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-reassembly mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially regulated Dvl dynamics.

Bindin is essential for fertilization in the sea urchin.

Species-specific sperm-egg interactions are essential for sexual reproduction. Broadcast spawning of marine organisms is under particularly stringent conditions, since eggs released into the water column can be exposed to multiple different sperm. Bindin isolated from the sperm acrosome results in insoluble particles that cause homospecific eggs to aggregate, whereas no aggregation occurs with heterospecific eggs. Therefore, Bindin is concluded to play a critical role in fertilization, yet its function has never been tested. Here we report that Cas9-mediated inactivation of the bindin gene in a sea urchin results in perfectly normal-looking embryos, larvae, adults, and gametes in both males and females. What differed between the genotypes was that the bindin-/- sperm never fertilized an egg, functionally validating Bindin as an essential gamete interaction protein at the level of sperm-egg cell surface binding.

Light-induced, spatiotemporal control of protein in the developing embryo of the sea urchin.

Differential protein regulation is a critical biological process that regulates cellular activity and controls cell fate determination. It is especially important during early embryogenesis when post-transcriptional events predominate differential fate specification in many organisms. Light-induced approaches have been a powerful technology to interrogate protein functions with temporal and spatial precision, even at subcellular levels within a cell by controlling laser irradiation on the confocal microscope. However, application and efficacy of these tools need to be tested for each model system or for the cell type of interest because of the complex nature of each system. Here, we introduce two types of light-induced approaches to track and control proteins at a subcellular level in the developing embryo of the sea urchin. We found that the photoconvertible fluorescent protein Kaede is highly efficient to distinguish pre-existing and newly synthesized proteins with no apparent phototoxicity, even when interrogating proteins associated with the mitotic spindle. Further, chromophore-assisted light inactivation (CALI) using miniSOG successfully inactivated target proteins of interest in the vegetal cortex and selectively delayed or inhibited asymmetric cell division. Overall, these light-induced manipulations serve as important molecular tools to identify protein function for for subcellular interrogations in developing embryos.

In silico determination of nitrogen metabolism in microbes from extreme conditions using metagenomics.

The acid ponds of the Danakil Depression in northern Ethiopia are polyextreme environments that exceed the normal physicochemical limits of pH, salinity, ion content, and temperature. We tested for the occurrence of DNA-based life in this environment using Metagenomic Shotgun DNA sequencing approaches. The obtained sequences were examined by the bioinformatic tools MetaSpades, DIAMOND and MEGAN 6-CE, and we were able to bin more than 90% of the metagenomics contigs of Dallol and Black Water to the Bacteria domain, and to the Proteobacteria phylum. Predictions of gene function based on SEED disclosed the presence of different nutrient cycles in the acid ponds. For this study, we focused on partial or completely sequenced genes involved in nitrogen metabolism. The KEGG nitrogen metabolism pathway mapping results for both acid ponds showed that all the predicted genes are involved directly or indirectly in the assimilation of ammonia and no dissimilation or nitrification process was identified. Furthermore, the deduced nitrogen fixation in the two acid ponds based on SEED classification indicated the presence of different sets of nitrogen fixing (nif) genes for biosynthesis and maturation of nitrogenase. Based on the in silico analysis, the predicted proteins involved in nitrogen fixation, especially the cysteine desulfurase and [4Fe-4S] ferredoxin, from both acid ponds are unique with less than 80% sequence similarity to the next closest protein sequence. Considering the extremity of the environmental conditions of the two acid ponds in the Danakil depression, this metagenomics dataset can add to the study of unique gene functions in nitrogen metabolism that enable thriving biocommunities in hypersaline and highly acidic conditions.